This application claims benefit of priority from U.S. provisional patent application serial No. 60/099,633, filed on Sep. 9, 1998. invention was funded in part by grant USDA-SBIR 97-03374 from the United States Department of Agriculture. The goverment has certain rights in this invention.
This invention relates to methods for generating doubled haploid plants from microspores, and to doubled haploid plants produced by the methods disclosed herein.
Although plant breeding programs worldwide have made considerable progress developing new cultivars with improved disease resistances, yields and other, useful traits, breeding as a whole relies on screening numerous plants to identify novel, desirable characteristics. Very large numbers of progeny from crosses often must be grown and evaluated over several years in order to select one or a few plants with a desired combination of traits.
In a typical plant breeding experiment, two parent plants are crossed and the resulting progeny (the F1 generation) are screened and a plant (termed the F1 plant) identified that possesses a desirable combination of phenotypic traits. The F1 plant is then self-fertilized to yield a population of progeny plants (termed F2 plants) that must be individually analyzed to determine which F2 plants possess the desired combination of phenotypic traits originally introduced in the F1 plant. If, as is often the case, the desired phenotypic traits derive from the combined effect of several genes, then the number of F2 progeny plants that must be screened depends on the number of genetic differences between the parents of the F1 plant. Thus, the greater the number of genetically-controlled differences between parents of the F1 plant, the larger the number of F2 progeny that must be grown and evaluated, and the lower the probability of obtaining progeny with all the desired traits.
For example, if the two parents of the F1 plant differ by 25 gene alleles (not an unusually great number in breeding), more land than exists on the earth would be needed to grow all possible genotype combinations which can occur in the F2 generation derived from the self-fertilized F1 plant (Konzak, C. F. et al. In: Elliott, L. (ed.) STEEPxe2x80x94Conservation Concepts and Accomplishment, pp. 247-273, 1987.). Further, once an F2 plant has been identified that exhibits the same, desirable, phenotypic trait(s) as the cross parents, the process of self-fertilization and analysis of the resulting progeny must be repeated several times until a homozygous population of plants is obtained which breed true for the desired phenotypic character, i.e., all progeny derived from the true-breeding population exhibit the desired, phenotypic trait (though the progeny may not be true-breeding for unselected traits).
One possible solution to the problem of screening large numbers of progeny is to produce them from the gametic cells as haploid plants, the chromosomes of which can be doubled using colchicine or other means to achieve instantly homozygous, doubled-haploid plants. In particular, doubled haploids can be produced from the microspores which normally give rise to pollen grains.
The life cycle of flowering plants exhibits an alteration of generations between a sporophytic (diploid) phase and a gametophytic (haploid) phase. Meiosis produces the first cells of the haploid generation which are either microspores (male) or megaspores (female). Microspores divide and develop within anthers to become mature male gametophytes (pollen). In normal development, microspores are genetically programmed for terminal differentiation to form mature pollen through two cell divisions. However, under certain conditions, microspores can be induced to initiate sporophytic development which leads to the formation of haploid or doubled haploid xe2x80x9cembryoidsxe2x80x9d. These embryoids can give rise to mature plants, that are either haploids or doubled haploids, through subsequent sporophytic development. The process by which plants are produced from microspores is termed pollen-embryogenesis or androgenesis, i.e., from the male gametophyte. Androgenesis is of significant interest for developmental genetic research as well as plant breeding and biotechnology, since it is a means to produce genetically true-breeding, doubled haploid plants.
As shown in Table 1, by producing doubled-haploid (also termed polyhaploid) progeny, the number of possible gene combinations for any number of inherited traits is more manageable.
Thus, marked improvements in the economics of breeding can be achieved via doubled haploid production, since selection and other procedural efficiencies can be markedly improved by using true-breeding (homozygous) progenies. With doubled haploid production systems, homozygosity is achieved in one generation. Thus, the breeder can eliminate the numerous cycles of inbreeding necessary by conventional methods to achieve practical levels of homozygosity. Indeed, true homozygosity for all traits is not even achievable by conventional breeding methods. Consequently, an efficient doubled haploid technology would enable breeders to reduce the time and the cost of cultivar development relative to conventional breeding practices.
Thus, there is a need for a method of efficiently producing doubled haploid plants that is applicable to a wide variety of plant species.
In accordance with the foregoing, in one aspect the present invention provides methods of generating doubled haploid and/or haploid plants from microspores.
The methods of the present invention for producing plants from microspores include the steps of: selecting plant material including microspores at a developmental stage amenable to androgenic induction; subjecting the microspores to temperature stress to obtain stressed microspores; contacting the microspores with an amount of a sporophytic development inducer effective to induce sporophytic development and chromosome doubling, the contacting step occurring before, during, after, or overlapping with any portion of the temperature stress step; isolating the stressed microspores; and coculturing the isolated microspores with either ovary-conditioned medium or at least one live plant ovary. Preferably, microspores are subjected to nutrient stress at the same time that they are subjected to temperature stress. Preferably, microspores are contacted with an amount of an auxin and/or a cell spindle inhibiting agent before, during, after, or overlapping with any portion of the temperature stress step.
In the practice of the methods of the present invention, plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction. Preferably the selected plant material is tillers or branches bearing spikes or flowers that contain microspores in the mid uninucleate to early binucleate stages of development. The microspores are treated by contacting the selected plant material with an aqueous medium, such as water, and subjecting the selected plant material to temperature stress, and optionally to nutrient stress. Temperature stress is effected by incubating the selected plant material, in contact with aqueous medium, at a preferred temperature of from about 4xc2x0 C. to about 40xc2x0 C., more preferably from about 28xc2x0 C. to about 35xc2x0 C., most preferably at about 33xc2x0 C., for a period of from about half an hour to about 72 hours. Nutrient stress is effected by utilizing, in the aqueous medium, an amount of at least one nutrient (such as nitrogen, calcium, phosphorus or sulfur) that is less than the amount of that nutrient necessary for the optimal growth and development of the microspores. Preferably nutrient stress is effected by utilizing water as the aqueous medium. Most preferably nutrient stress is effected by utilizing diluted NPB 98 as the aqueous medium, preferably NPB 98 medium diluted with an amount of water sufficient to dilute NPB 98 to less than or equal to 80% of its undiluted concentration. The selected plant material is also contacted with an effective amount of at least one sporophytic development inducer (as further described herein), such as 2-hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel. Preferably the selected plant material is contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin (preferably 2,4-dichlorophenoxyacetic acid) and/or an effective amount of a cell spindle inhibiting agent (such as pronamide). The presently preferred concentration range for auxin is from about 0.1 mg/l to about 25 mg/l, more preferably from about 0.2 mg/l to about 10.0 mg/l, most preferably from about 0.5 mg/l to about 4.0 mg/l. The presently preferred concentration range for sporophytic development inducer is from about 0.001 mg/l to about 1000 mg/l. The presently most preferred concentration range for sporophytic development inducer is from about 1 mg/l to about 500 mg/l. The presently preferred concentration range for cell spindle inhibiting agent is from about 1.0 xcexcM to about 200 xcexcM.
Optionally, the selected plant material is contacted with an effective amount of a cytokinin, preferably kinetin or BAP, and/or an effective amount of a gibberellin. The preferred concentration range for cytokinin is from about 0.1 mg/l to about 10 mg/l, more preferably from about 0.2 mg/l to about 4.0 mg/l, most preferably from about 0.5 mg/l to about 2.0 mg/l. The presently preferred concentration range for gibberellin is from about 0.01 mg/l to about 20 mg/l, most preferably from about 0.2 mg/l to about 4.0 mg/l. The selected plant material is contacted with some or all of the foregoing chemical agents (sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin) before, during, after, or overlapping with any portion of the temperature stress treatment. The treated microspores are isolated preferably by macerating the selected, treated plant tissue, filtering the macerated plant tissue and subjecting the filtrate to density centrifugation, preferably utilizing a solution of percoll, ficoll or mannitol, most preferably a 0.3 M mannitol solution, layered over a higher density solution of percoll, ficoll, polyethylene glycol or a sugar, preferably maltose, most preferably 0.58 M maltose. The isolated, treated microspores are then cultured in a liquid nutrient suspension medium, such as medium NPB98 or NPB 99, preferably NPB 99, supplemented with either plant ovary conditioned medium or at least one live plant ovary, until the microspores develop into embryoids. Preferably the plant ovaries (including the ovaries used to prepare plant ovary conditioned medium) are obtained from wheat varieties xe2x80x9cChrisxe2x80x9d or xe2x80x9cPavon 76xe2x80x9d, but ovaries from a wide range of genotypes, including Igri barley, are effective. The embryoids are transferred to a regeneration medium and incubated therein until the embryoids develop into plants. The resulting plants may be doubled haploids, or they may be haploids which can be converted to doubled haploids by treatment with a chromosome doubling agent such as colchicine. It will be understood, however, that the microspores can be isolated before being contacted with an aqueous medium and being subjected to temperature stress.
The methods of the present invention for producing plants from microspores may optionally include the step of genetically transforming the microspores. Microspores can be genetically transformed at any time during treatment of the microspores in accordance with the methods of the present invention. The presently preferred methods of genetically transforming microspores are biolistic gene transfer utilizing a particle gun or electroporation of plasmolyzed microspores. Thus, in one aspect, the present invention provides genetically transformed plants regenerated from microspores.
In other aspects, the present invention provides methods of initiating microspore embryogenesis including the steps of: selecting plant material including microspores at a developmental stage amenable to androgenic induction; subjecting the microspores to temperature stress to obtain stressed microspores; and contacting the microspores with an amount of a sporophytic development inducer effective to induce sporophytic development and chromosome doubling, the contacting step occurring before, during, after, or overlapping with any portion of the temperature stress step. Preferably, microspores are subjected to nutrient stress at the same time that they are subjected to temperature stress. Preferably, microspores are contacted with an amount of an auxin and/or a cell spindle inhibiting agent before, during, after, or overlapping with any portion of the temperature stress step.
In the practice of the methods of the present invention for initiating microspore embryogenesis, plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction. Preferably the selected plant material is tillers or branches bearing spikes or flowers that contain microspores in the mid uninucleate to early binucleate stages of development. The microspores are treated by contacting the selected plant material with an aqueous medium, such as water, and subjecting the selected plant material to temperature stress, and optionally to nutrient stress. Temperature stress is effected by incubating the selected plant material, in contact with aqueous medium, at a preferred temperature of from about 4xc2x0 C. to about 40xc2x0 C., more preferably from about 28xc2x0 C. to about 35xc2x0 C., most preferably at about 33xc2x0 C., for a period of from about half an hour to about 72 hours. Nutrient stress is effected by utilizing, in the aqueous medium, an amount of at least one nutrient that is less than the amount of that nutrient necessary for the optimal growth and development of the microspores. Preferably nutrient stress is effected by utilizing water as the aqueous medium. Most preferably nutrient stress is effected by utilizing diluted NPB 98 as the aqueous medium, preferably NPB 98 medium diluted with an amount of water sufficient to dilute NPB 98 to less than or equal to 80% of its undiluted concentration. The selected plant material is preferably subjected to nutrient stress for a period of from about half an hour to about ninety six hours, more preferably from about half an hour to about seventy two hours. The selected plant material is also contacted with an effective amount of at least one sporophytic development inducer (as further described herein), such as 2-hydroxynicotinic acid (2-HNA), violuric acid, 2-hydroxyproline or ethrel. Preferably the selected plant material is contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin (preferably 2,4-dichlorophenoxyacetic acid) and/or an effective amount of a cell spindle inhibiting agent (such as pronamide). The presently preferred concentration range for auxin is from about 0.1 mg/l to about 25 mg/l, more preferably from about 0.2 mg/l to about 10.0 mg/l, most preferably from about 0.5 mg/l to about 4.0 mg/l. The presently preferred concentration range for sporophytic development inducer is from about 0.001 mg/l to about 1000 mg/l. The presently most preferred concentration range for sporophytic development inducer is from about 1 mg/l to about 500 mg/l. The presently preferred concentration range for cell spindle inhibiting agent is from about 1.0 xcexcM to about 200 xcexcM.
Optionally, the selected plant material is contacted with an effective amount of a cytokinin, preferably kinetin or BAP, and/or an effective amount of a gibberellin. The preferred concentration range for cytokinin is from about 0.1 mg/l to about 10 mg/l, more preferably from about 0.2 mg/l to about 4.0 mg/l, most preferably from about 0.5 mg/l to about 2.0 mg/l. The presently preferred concentration range for gibberellin is from about 0.01 mg/l to about 20 mg/l, most preferably from about 0.2 mg/l to about 4.0 mg/l. The selected plant material is contacted with some or all of the foregoing chemical agents (sporophytic development inducer, cell spindle inhibiting agent, auxin, cytokinin and/or gibberellin) before, during, after, or overlapping with any portion of the temperature stress treatment. It is understood, however, that the microspores can be isolated before being contacted with an aqueous medium and being subjected to temperature stress.
The methods of the present invention for initiating microspore embryogenesis may optionally include the step of genetically transforming the microspores. Preferably uninucleate microspores are used for genetic transformation. Microspores can be genetically transformed at any time during treatment of the microspores in accordance with the methods of the present invention. The presently preferred methods of genetically transforming microspores are biolistic gene transfer utilizing a particle gun or electroporation of plasmolyzed microspores.
In another aspect of the invention, methods are provided for stimulating susceptible plant microspores to form embryoids. The methods include the step of incubating susceptible microspores with at least one whole plant ovary, or with plant ovary conditioned medium capable of stimulating susceptible plant microspores to form embryoids.
In another aspect of the present invention, doubled haploid and/or haploid plants are provided that are produced according to the methods of the present invention.